Effect of compounds that suppress induction of plasminogen activator inhibitor 1 (PAI-1)

ABSTRACT

The present invention provides methods of inhibiting PAI-1 using benzopyrans.

This application claims the benefit of U.S. Provisional Application No. 60/022308, filed Jul. 15, 1996.

BACKGROUND OF THE INVENTION

The fibrinolytic system plays a key role in maintaining normal hemostatic balance. A critical factor in this system is plasminogen activator inhibitor I (PAI-1), which reduces the endogenous ability to remove fibrin by inhibiting plasminogen activators such as tissue type plasminogen activator (tPA). Studies have documented that elevations of PAI-1 are associated with increased risk of deep venous thrombosis. Further, elevations in PAI-1 are found in patients suffering from myocardial infarction and septicemia. Because impaired fibrinolytic capacity is associated with increased cardiovascular risk, lowering PAI-1 should result in cardioprotection. In fact, recent studies on the analysis of PAI-1 levels in pre- and post-menopausal women in the Framingham Offspring Study have demonstrated that post-menopausal women have markedly higher PAI-1 levels, which can be reduced to pre-menopausal levels with estrogen therapy. This reduction in PAI-1 effect is believed to contribute to the overall effect of estrogen replacement therapy on the reduced risk of heart disease.

While PAI-1 can be produced in a variety of tissues, substantial levels are secreted by the vascular endothelial cell. The vascular endothelium constitutes a major organ that functions in the regulation of blood coagulation, inflammation and in the exchange of fluids and mediators between the intravascular compartment and parenchyma tissues. As such, the proper function of the endothelium is critical to overall homeostasis. Because PAI-1-can be increased in endothelial cells in response to certain stimuli, including cytokines, it contributes to a dysfunctional state that can result in coagulation defects, local and systemic vascular inflammation, and enhancement in the progression and rupture of atherosclerotic plaque. These effects can further result in conditions including myocardial infarction, deep venous thrombosis, and disseminated intravascular thrombosis.

Because the local control of PAI-1 at the endothelial cell/plasma interface can play a major role in many pathological processes, agents that inhibit the expression of PAI-1 in the endothelium could be useful in treating or preventing conditions such as sepsis, injuries involving major tissue damage and trauma, systemic inflammatory response syndrome, sepsis syndrome, septic shock and multiple organ dysfunction syndrome (including DIC) as well as myocardial infarction, deep venous thrombosis, disseminated intravascular thrombosis, atherosclerotic plaque rupture and its associated sequela.

In addition, tPA (tissue Plasiminogen Activator) is currently administered to patients who have suffered from conditions which place them at risk of detrimental thrombotic events. Exogenously administered tPA has been shown to be effective and is commercially available for treatment of such patients. However, efficacy with this therapy can be limited because PAI-1 inhibits the exogenously given tPA as well as the endogenously derived tPA. Therefore, it would be of great value if an agent were available which could either prolong the half-life or reduce the amount of exogenously administered tPA.

Further, because of the critical role of fibrin in tumor cell biology, agents that modulate PAI-1 may find use as anti-metastatic agents.

SUMMARY OF THE INVENTION

This invention provides methods for inhibiting plasminogen activator inhibitor 1 comprising administering to a human in need thereof an effective amount of a compound of formula I ##STR1##

wherein R¹ and R² are, independently, --OH, --O(CO)C₁ -C₆ alkyl, --O(CO)phenyl or substituted phenyl, --O(CO)O(C₁ -C₆ alkyl), or --O(CO)O-phenyl;

R³ is ##STR2## and

R⁴ is --H, methyl, ethyl, propyl, ethenyl or ethynyl; or a pharmaceutically acceptable salt or solvate thereof.

Also provided by the invention are articles of manufacture comprising compounds of formula I.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns the discovery that a select group of 2-phenyl-3-aryl-dihydrobenzopyrans, those of formula I, are useful for inhibiting PAI-1.

The methods of use provided by this invention are practiced by administering to a human in need thereof a dose of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, that is effective to inhibit PAI-1 or a physiological condition associated with an excess thereof. The term "inhibit" includes its generally accepted meaning which includes prohibiting, preventing, restraining, and slowing, stopping, or reversing progression, severity, or ameliorating a resultant symptom or effect.

General terms used in the description of compounds herein described bear their usual meanings. For example, "C₁ -C₆ alkyl" refers to straight or branched aliphatic chains of 1 to 6 carbon atoms including methyl, ethyl, propyl, iso-propyl, n-butyl, pentyl, iso-pentyl, hexyl, and the like.

The term "substituted phenyl" refers to a phenyl group having one or more substituents selected from the group consisting of C₁ -C₄ alkyl, C₁ -C₃ alkoxy, hydroxy, nitro, chloro, fluoro, or tri(chloro or fluoro)methyl. "C₁ -C₃ alkoxy" refers a C₁ -C₃ alkyl group attached through an oxygen bridge such as, methoxy, ethoxy, n-propoxy, iso-propoxy.

All of the compounds used in the methods of the current invention can be made according to established or analogous procedures, such as those detailed in European Patent Application No. 0470310A1 and PCT Application WO 93/10741, both of which are incorporated herein by reference. In addition, compounds germane to the current invention may be made by procedures shown in U.S. Pat. No. 5,446,061, which is also incorporated by reference. Modifications to these methods may be necessary to accommodate reactive functionalities of particular substituents. Such modifications are either apparent to, or readily ascertained by, those skilled in the art of organic chemistry.

Compounds used in the invention include:

2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-7-hydroxy-2H-1-benzopyran hydrochloride

2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-acetyloxyphenyl)-7-acetyloxy-2H-1-benzopyran hydrochloride

2-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-3-(4-hydroxyphenyl-7-hydroxy-2H-1-benzopyran

2-[4-[2-(1-pyrrolidino)ethoxy]phenyl]-3-(4-benzoyloxyphenyl)-7-benzoyl-2H-1-benzopyran

2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-4-methyl-7-hydroxy-2H-1-benzopyran

2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-4-ethyl-7-hydroxy-2H-1-benzopyran

A preferred embodiment of this invention is 2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-4-methyl-7-hydroxy-2H-1-benzopyran.

The compounds used in the methods of this invention form pharmaceutically acceptable acid salts with a wide variety of organic and inorganic acids and include the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like. Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like. A preferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene. The salt normally precipitates out of solution within about one hour to 10 days and can be isolated by filtration or the solvent can be stripped off by conventional means.

The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.

Specific examples of the compounds of formula I include but are not limited to the following:

Pharmaceutical formulations can be prepared by procedures known in the art. For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes. Additionally, the compounds are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances or waxes.

The particular dosage of a compound of formula I required to inhibit PAI-1, or any other use disclosed herein, and according to this invention will depend upon the severity of the condition, the route of administration, and related factors that will be decided by the attending physician. Generally, accepted and effective daily doses will be from about 0.1 to about 1000 mg/day, and more typically from about 50 to about 200 mg/day. Such dosages will be administered to a subject in need thereof from once to about three times each day, or more often as needed to effectively inhibit PAI-1, or any other use disclosed herein.

Formulations

In the formulations which follow, "Active ingredient" means a compound of formula I.

Formulation 1

Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Active ingredient 0.1-1000                                                       Starch, NF 0-650                                                               Starch flowable powder 0-650                                                   Silicone fluid 350 centistokes 0-15                                          ______________________________________                                    

The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules.

The specific formulations above may be changed in compliance with the reasonable variations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2

Tablets

    ______________________________________                                         Ingredient       Quantity (mg/tablet)                                          ______________________________________                                         Active ingredient                                                                               0.1-1000                                                        Cellulose, microcrystalline 0-650                                              Silicon dioxide, fumed 0-650                                                   Stearate acid 0-15                                                           ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.1-1000 mg of active ingredient are made up as follows:

Formulation 3

Tablets

    ______________________________________                                         Ingredient         Quantity (mg/tablet)                                        ______________________________________                                         Active ingredient Compound 5                                                                      80                                                            Starch 45                                                                      Cellulose, microcrystalline 35                                                 Polyvinylpyrrolidone 4                                                         (as 10% solution in water)                                                     Sodium carboxymethyl cellulose 4.5                                             Magnesium stearate 0.5                                                         Talc 1                                                                       ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose are made as follows:

Formulation 4

Suspensions

    ______________________________________                                         Ingredient         Quantity (mg/5 ml)                                          ______________________________________                                         Active ingredient  0.1-1000     mg                                               Sodium carboxymethyl cellulose 50 mg                                           Syrup 1.25 mg                                                                  Benzoic acid solution 0.10 mL                                                Flavor             q.v.                                                          Color q.v.                                                                   Purified water to  5            mL                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.

To demonstrate the utility for the compounds of formula I in inhibiting PAI-1, the following experimental procedure was performed.

Endothelial Cell PAI-1 Assay

96 well tissue culture plates were prepared with 1×10⁴ human endothelial cells (HUVEC) per well in Clonetics' Endothelial Cell Growth Medium (EGM) supplemented with 2% FBS. Following incubation overnight at 37₋₋ C., the medium was replaced with serum-free medium (DMEM/F-12 medium, 20 mM-HEPES, pH 7.5, 50 μg/ml gentamicin, 1 μg/ml human transferrin and 1 μg/ml bovine insulin) with or without compound 1, (where R¹ and R² are hydroxy, and R³ is ##STR3## and with or without 1 nM IL-1 beta. Following incubation overnight at 37₋₋ C., samples of culture medium were assayed for secreted PAI-1 using the Imubind Plasma PAI-1 ELISA (American Diagnostic Inc. #822/1S).

Results

Human umbilical vein endothelial cells (HUVEC) were treated with compound 1 concurrent to the induction of PAI-1 with IL-1. In initial experiments with several lots of cells obtained from a commercial supplier (Clonetics), we found that not all lots were responsive to 17 beta estradiol, and were thus not used in experiments to determine the effect of compound 1 on PAI-1 secretion. As shown in Table 1, using an estrogen-responsive line, we observed that compound 1 (A preferred embodiment of this invention is 2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-4-methyl-7-hydroxy-2H-1-benzopyran) significantly reduced the induction of PAI-1 by IL-1-at a concentration of 1 nM. These data demonstrate that compound 1 is a potent inhibitor of the induction of PAI-1 from activated endothelial cells and should result in a cardioprotective effect, i.e. reduction in the incidence of cardiovascular events, due to enhancing fibrinolytic potential. Further the positive effect of compound 1 on reducing PAI-1 may provide for acute and chronic uses in conditions where elevated levels are associated with pathology or may be used to prevent such pathological conditions.

                  TABLE 1                                                          ______________________________________                                         Effect of compound 1 on PAI-1 secretion from human                               endothelial cells                                                                                 PAI-1 Induction                                             Treatment % of IL-1 Control*                                                 ______________________________________                                         IL-1             100                                                             IL-1 & 1nM compound 1 58 +/-4                                                  IL-1 & 10nM compound 1 12 +/-6                                               ______________________________________                                          *(drug treated  control)/(Il1 treated  control) × 100%              

We claim:
 1. A method of inhibiting plasminogen activator inhibitor or a physiological condition associated with an excess thereof, comprising administering to a human in need thereof an effective amount of a compound having the formula ##STR4## wherein R¹ and R² are, independently, --OH, --OCO(C₁ -C₆ alkyl), --O(CO)phenyl or substituted phenyl, --O(CO)O(C₁ -C₆ alkyl), or --O(CO)O-phenyl;R³ is ##STR5## and R⁴ is --H, methyl, ethyl, propyl, ethenyl or ethynyl;or a pharmaceutically acceptable salt or solvate thereof.
 2. The method of claim 1 wherein said compound is 2-[4-[2-(1-piperidino)ethoxy]phenyl]-3-(4-hydroxyphenyl)-4-methyl-7-hydroxy-2H-1-benzopyran.
 3. An article of manufacture comprising packaging material and a pharmaceutical formulation contained within said packaging material, wherein said pharmaceutical formulation is effective for inhibiting plasminogen activator inhibitor or a physiological condition associated with an excess thereof, wherein said packaging material comprises a label which indicates said pharmaceutical formulation can be used for inhibiting plasminogen activator inhibitor or a physiological condition associated with an excess thereof, and wherein said pharmaceutical formulation comprises a compound of formula I ##STR6## wherein R¹ and R² are, independently, --OH, --OCO(C₁ -C₆ alkyl), --O(CO)phenyl or substituted phenyl, --O(CO)O(C₁ -C₆ alkyl), or --O(CO)O-phenyl;R³ is ##STR7## and R⁴ is --H, methyl, ethyl, propyl, ethenyl or ethynyl;or a pharmaceutically acceptable salt or solvate thereof. 